The AIDS opportunistic pathogen, Cryptococcus neoformans, produces large amounts of the 6-carbon acyclic polyol mannitol in culture and infected animals. This project's long-term goal is to ascertain the functional and pathogenic significance of the metabolic pathways by which C. neoformans synthesizes and catablizes mannitol. Aim 1 is to elucidate the mannitol biosynthetic pathway and to assess this pathway s significance. Mannitol-1-phosphate dehydrogenase (MPD) catalyzes a key step in mannitol biosynthesis in other fungi, and we have cloned a C. neoformans cDNA encoding MPD. Therefore, the MPD structural gene will be cloned and disrupted, and the resulting null mutants will be used to determine if the ability to synthesize mannitol is required for wild-type stress tolerance and virulence. Aim 2 is to elucidate the mannitol catabolic pathway and to assess this pathway s significance. NAD- and NADP-dependent mannitol dehydrogenases (NAD-MDH and NADP-MDH, respectively) catalyze key steps in mannitol catabolism in other fungi, and lysates of C. neoformans contain both enzymes. Therefore, the genes encoding NAD-MDH and NADP-MDH will be cloned and disrupted, and the resulting null mutants will be tested for stress tolerance and virulence. Aim 3 is to examine the roles of MPD, NAD-MDH and NADP-MDH in regulating mannitol biosynthesis, catabolism and accumulation. Total and intracellular mannitol will be measured when C. neoformans is subjected to various environmental conditions, and the results will be correlated with MPD, NAD-MDH and NADP-MDH mRNA and protein levels. Also, the functions of a C. neoformans gene (MTL1) that permits Saccharomyces cerevisiae to express a cryptic NAD-MDH will be studied by overexpressing MTL1 in C. neoformans and by constructing and analyzing mtl1 null mutants.